Conus is a genus of predatory marine gastropods (snails) which envenomate their prey. Venomous cone snails use a highly developed injection apparatus to deliver their cocktail of toxic conotoxins into their prey. In fish-eating species such as Conus magus, the cone detects the presence of the fish using chemosensors in its siphon and, when close enough, extends its proboscis and impales the fish with a hollow harpoon-like tooth and injects the venom into the fish. This injection immobilizes the fish and enables the cone snail to wind it into its mouth via the proboscis. For general information on Conus and their venom, see Coleman, N. (2nd ed., 1992. Ure Smith Press, Sydney, Australia (ISBN 0 7254 0885 5)).
Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes. Each Conus specie's venom appears to contain a unique set of 50–200 peptides. The composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyze its prey. The active components of the venom are small peptide toxins, typically 12–30 amino acid residues in length, and are typically highly constrained peptides due to their high density of disulfide bonds.
The venoms consist of a large number of different peptide components that, when separated, exhibit a range of biological activities. When injected into mice, they elicit a range of physiological responses from shaking to depression. The paralytic components of the venom that have been the focus of recent investigation are the α-, ω- and μ-conotoxins. All of these conotoxins are believed to act by preventing neuronal communication, but each targets a different aspect of the process to achieve this action. Each venom component has a very specific pharmacologic target. For example, a linkage has been established between α-, αA- and ψ-conotoxins and the nicotinic ligand-gated ion channel; ω-conotoxins and the voltage-gated calcium channel; μ-conotoxins and the voltage-gated sodium channel; δ-conotoxins and the voltage-gated sodium channel; κ-conotoxins and the voltage-gated potassium channel; conantokins and conodynes and the ligand-gated glutamate (NMDA) channel. (Olivera et al., 1985; Olivera et al., 1990.) The pharmacological specificity of the conotoxins makes them attractive for drug development for a variety of therapeutic applications, including neurological and cardiovascular disorders.
A characteristic structural feature of conotoxins is a large number of posttranslational modifications, in particular disulfide bridges. The primary function of disulfide bonds appears to be stabilization of the structure. Conotoxins are grouped into families, based upon the number and arrangement of disulfide bonds. For example, two disulfide-containing α-conotoxins contain the cysteine pattern, CC—C—C, with disulfides between the 1st and 3rd, 2nd and 4th cysteines. Three disulfide-containing ω- and δ-conotoxins share the native cysteine pattern, C—C—CC—C—C, whereas μ-conotoxins share the common cysteine pattern, CC—C—C—CC. For native ω-, δ-conotoxins, the 1st and 4th, 2nd and 5th and 3rd and 6th cysteines are connected; for native μ-conotoxins, the 1st and 4th, 2nd and 5th and 3rd and 6th cysteines are connected by disulfide bonds. The correct pairing of disulfides in the native conotoxins is a prerequisite for maintaining their biological activity. The disulfide bridges are formed in a process of oxidative pairing of the cysteine residues.
Conotoxins are naturally synthesized as precursors in cells (Woodward et al., 1990; Colledge et al., 1992). For all conotoxins, the precursors share a similar organization: an N-terminal signal sequence, a propeptide region and a C-terminal cysteine-rich toxin region. Each family of conotoxins is characterized by a highly conserved signal sequence, a moderately conserved propeptide region and an almost random toxin region that contains a conserved cysteine framework.
Propeptides have been shown in many biological systems to assist in the oxidative folding of polypeptides. Examples of those studies are summarized in Table 1. Folding kinetics and yields can be significantly improved when oxidation of cysteine-rich peptides is carried out using the propeptide. In the case of proguanylin, a peptide containing two disulfide bridges, folding yields improved from 7%, using mature peptide, to 95%, using the propeptide (Schulz et al., 1999). Similar studies on the guanylyl cyclase-activating peptide, GCAP-II, showed that two amino acids in the N-terminal fragment of the propeptide were directly involved in the enhancement of peptide folding (Hidaka et al., 2000). For oxidative folding of bovine pancreatic trypsin inhibitor (BPTI), the propeptide substantially increased the folding yields and the kinetics of folding through an additional N-terminal cysteine residue present in the propeptide fragment. It thus appears that propeptides can facilitate oxidative folding of polypeptides.
TABLE 1Summary of intramolecular and intermolecular factors influencing theoxidative folding of polypeptides.FactorsExamples of polypeptidesPropeptide-assistedMacrophage inhibitory cytokine-1 MIC-1 (Fairlie et al., 2001), Nerveoxidative foldinggrowth factor hNGF (Rattenholl et al., 2001), Prouroguanylin, GCAP(Hidaka et al., 1998; Schulz et al., 1999; Hidaka et al., 2000), pancreatictrypsin inhibitor BPTI (Weissman and Kim, 1992)ChaperonesHsp70 - binding to early folding intermediates (BiP/GRP78, GRP170),Hsp70/hsp40Hsp40 - cochaperones regulating Hsp70 (Sec63p, DnaJ), Hsp90 -Calreticulin/calnexingeneral chaperones (GRP94), Hsp25 (small heat-shock proteins withsingle Cys residues), Lectins - quality control of folding (calnexin,calreticulin) immunophilins - isomerization of prolines (cyclophilin,FKPB13) (Gething, 1997; 1999)Disulfide isomerasesPDI (Freedman et al., 1994; Gilbert, 1997)and otherErp72, CaPB1, CaPB2 (Rupp et al., 1994)oxido-reductasesEro1p (Tu et al., 2000)Erv2 (Servier et al., 2001)
However, not all propeptides have been shown to increase the folding yields and/or the kinetics of folding. For example, ω-conotoxin MVIIA and insulin-like growth factor (IGF) are two reported examples where a propeptide did not have a direct effect on oxidative folding. Studies by Price-Carter and Goldenberg (Price-Carter et al., 1996b) suggested that the propeptide sequence neither increased folding yields nor enhanced the kinetics of folding of ω-MVIIA. While, mature ω-MVIIA folds with relatively high yields, using the propeptide of IGF did not facilitate folding. In the case of IGF, the propeptide, likewise, did not facilitate folding.
Taken together, these studies demonstrate examples where the propeptide is very important in determining folding properties of polypeptides, as well as examples where a propeptide is not directly involved in the folding mechanism. In addition to the possible role played by propeptides, a number of other molecules are known to regulate the folding pathway of peptides in order to increase the kinetics and yields of properly folded forms.
Molecular chaperones comprise a large number of proteins that are specialized as folding assistants. Their general function is to prevent the aggregation and precipitation of nascent polypeptides and folding intermediates. These chaperones are localized in the cytoplasm and in the endoplasmic reticulum (“ER”) and bind to different folding species with relatively low specificity. The ER is the main protein-folding compartment where a majority of chaperones are involved in folding, quality control and translocation of polypeptides. Since the ER is also the only compartment where oxidative folding occurs, chaperones in the ER play a prominent role in the oxidative folding of proteins. For example, BiP, a member of the Hsp70 chaperone family, was recently shown to cooperate with protein disulfide isomerase in the oxidative folding of antibodies (Mayer et al., 2000). Some examples of molecular chaperones are summarized in Table 1.
The oxidative folding of polypeptides in vivo is catalyzed by protein disulfide isomerase (PDI), which can act as both a folding catalyst and as a molecular chaperone. The activity of this enzyme was originally discovered in rat liver, but since then it has been documented in a variety of different species. PDI belongs to a group of protein-thiol oxidoreductase enzymes, which contain thioredoxin domains. A typical PDI molecule consists of two similar thioredoxin-like domains. These domains contain the Cys-Gly-His-Cys (CGHC) (SEQ ID NO:19) redox active site. The C-terminal region of PDI has an additional domain with an ER retention signal sequence. However, there are many different classes of PDIs which are distinguished based upon their thioredoxin domain arrangement and composition as summarized in (McArthur, A. G. et al., Mol. Biol. Evol. 18(8) 1455-63, 2001).
PDI catalyzes protein thiol-disulfide exchange reactions using the thioredoxin CGHC (SEQ ID NO:19) redox active site. The enzyme contains two CGHC (SEQ ID NO:19) motifs, a low affinity peptide binding site and a KDEL (SEQ ID NO:20) endoplasmic reticulum retrieval signal. PDI is also characterized by a large number of low affinity/high capacity calcium binding sites. The oxidoreductase activity of PDI is mediated by the pair of Cys residues in the active site. These Cys residues can be easily reduced to thiols, or oxidized to a disulfide, depending on the redox potential and relative concentration of substrates and products in the ER. Moreover, PDI was also shown to be sufficient for promoting oxidative folding, even in the absence of glutathione, a molecule primarily responsible for maintaining the oxidative environment of the ER (Tu et al., 2000). In addition to its catalytic role in oxidative folding, PDI can also function as a molecular chaperone. PDI was found to facilitate folding of proteins lacking disulfides, such as rhodanase or glyceraldehyde-3-phosphate dehydrogenase. This dual function of PDI was recently characterized during the oxidative folding of proinsulin (Winter et al., 2002). In the proinsulin study, PDI increased the rate of oxidative folding and prevented proinsulin aggregation.
Since PDI has been found in bacteria, fungi, plants, invertebrate and vertebrate animals, we sought to determine if Conus snails have also utilized this enzyme to produce conotoxins. Because Conus species produce a large number of disulfide-rich proteins in their venom, a need exists in the art to identify the nucleic acid sequences encoding Conus protein disulfide isomerases, to identify the sequences of Conus protein disulfide isomerases, and to use the nucleic acids or proteins in the folding of disulfide-containing proteins.